Being first reported in 2001, store-operated Ca2+ entry (SOCE) is a relatively new phenomenon in skeletal muscle. SOCE is coordinated by coupling between two proteins: STIM1 calcium sensors in the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the transverse tubule (TT) membrane. SOCE enhances muscle growth/development, limits fatigue, and promotes fatigue-resistant type I fiber specification. On the other hand, SOCE dysfunction contributes to muscle weakness/fatigue in aging, exacerbates muscular dystrophy, and mutations in STIM1 and Orai1 genes result in debilitating myopathies. The picture that emerges is that tight regulation of STIM1/Orai1-dependent SOCE activity is critical for optimal muscle performance such that increases or decreases in SOCE activity can lead to muscle fatigue, sarcopenia, and myopathy. While SOCE activity clearly impacts muscle performance, sites of STIM1-Orai1 coupling in muscle remain unclear. For this renewal, we developed inducible, muscle-specific Orai1 and STIM1 KO mice to determine the mechanism by which STIM1-Orai1 coupling limits fatigue. We provide exciting evidence that fatiguing exercise drives the formation of heretofore undescribed junctions between the TT and SR where STIM1-Orai1 coupling occurs, which we refer to as Ca2+ entry units (CEUs). CEUs are connected to, but distinct from, the triad or Ca2+ release unit. We provide preliminary data that Orai1 has a stronger impact on muscle fiber contractile function in female compared to male mice. We also provide preliminary collaborative immunoprecipitation and mass spectroscopy feasibility data for characterizing the STIM1 proteome before and after CEU formation. We will use these research tools, approaches, discoveries, and collaborations to advance understanding of the molecular determinants, subcellular location, and functional role of SOCE in skeletal muscle. Based on our published and preliminary data, we hypothesize that fatiguing exercise triggers formation of junctional extensions of the triad containing activated STIM1-Orai1 complexes that coordinate SOCE to enhance SR calcium refilling, limit muscle fatigue, and over the long-term, promote NFATc1 nuclear localization and type I fiber specification. We also hypothesize that fatigue-induced CEU formation in muscle involves a complex coordination of multiple protein components (including Bin1, STIM1, and Orai1). We propose to test these hypotheses according to the following two Specific Aims. Aim 1 will characterize the role of Orai1 in muscle fatigue and Type I fiber specification. Aim 2 will identif the subcellular location, molecular components, and stability of newly identified CEUs in adult skeletal muscle and determine the dependence of CEU formation and disassembly on the development of and recovery from fatigue. This project will: 1) provide novel mechanistic insights into the physiological role and subcellular location of SOCE in muscle, 2) use targeted and non-biased discovery approaches to identify and validate proteins involved CEU formation, and 3) determine the impact of gender on Orai1-dependent SOCE function, fatigue, fiber type specification, and CEU formation.